CD19 CAR T cells for B cell malignancies: a systematic review and meta-analysis focused on clinical impacts of CAR structural domains, manufacturing conditions, cellular product, doses, patient’s age, and tumor types

CD19-targeted chimeric antigen receptors (CAR) T cells are one of the most remarkable cellular therapies for managing B cell malignancies. However, long-term disease-free survival is still a challenge to overcome. Here, we evaluated the influence of different hinge, transmembrane (TM), and costimulatory CAR domains, as well as manufacturing conditions, cellular product type, doses, patient’s age, and tumor types on the clinical outcomes of patients with B cell cancers treated with CD19 CAR T cells. The primary outcome was defined as the best complete response (BCR), and the secondary outcomes were the best objective response (BOR) and 12-month overall survival (OS). The covariates considered were the type of hinge, TM, and costimulatory domains in the CAR, CAR T cell manufacturing conditions, cell population transduced with the CAR, the number of CAR T cell infusions, amount of CAR T cells injected/Kg, CD19 CAR type (name), tumor type, and age. Fifty-six studies (3493 patients) were included in the systematic review and 46 (3421 patients) in the meta-analysis. The overall BCR rate was 56%, with 60% OS and 75% BOR. Younger patients displayed remarkably higher BCR prevalence without differences in OS. The presence of CD28 in the CAR’s hinge, TM, and costimulatory domains improved all outcomes evaluated. Doses from one to 4.9 million cells/kg resulted in better clinical outcomes. Our data also suggest that regardless of whether patients have had high objective responses, they might have survival benefits from CD19 CAR T therapy. This meta-analysis is a critical hypothesis-generating instrument, capturing effects in the CD19 CAR T cells literature lacking randomized clinical trials and large observational studies. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-024-12651-6.


Introduction
Chimeric antigen receptors (CARs) are artificial cell membrane receptors responsible for immune cell activation.They are constituted by an extracellular binding domain selected against an antigen, usually in the form of a single-chain variable fragment (scFv), a hinge sequence, and a transmembrane domain fused to intracellular costimulatory and stimulatory signaling domains.First-generation CARs had only one CD3ζ chain in the intracellular domain for T cell activation.Second-and third-generation CARs harbor one and two additional intracellular costimulatory domains, respectively, eliciting complete T cell activation.Fourth-generation CARs are based on second or third-generation CARs designed in a vector able to induce the expression of additional transgenic products, constitutively or by induction, such as cytokines or monoclonal antibodies.The CAR expression has been vastly explored in T cells (CAR T cells), and is evolving in other immune cell types, such as NK cells, dendritic cells, and macrophages, ushering in a new era for the treatment of cancer and other diseases [1,2].In clinical trials, the main domains constituting the hinge part of a CAR are CD28, CD8 alpha, IgG4, or IgG1, while for the transmembrane domain (TM), CD28 or CD8 alpha are the most applied.The costimulatory domains more extensively applied in the clinical setting are CD28 and 4-1BB.CD28 incorporation into the costimulatory domain of CD19 CAR elicits tumor eradication, glycolysis, effector memory maturation, and T cell exhaustion, whereas 4-1BB signaling induces in vivo T cell persistence, mitochondrial biogenesis, and reprogramming towards a central memory T cell phenotype [3].Regardless of a few small studies that explored the clinical impact of using different costimulatory domains in the CAR, there is a lack of information about the influence of different hinge or TM domains on the clinical outcomes of patients treated with CAR T cells.
One of the current most effective CAR T cell therapies targets CD19, an antigen expressed by B cells in all stages of development until differentiation in plasmocytes, including B cell malignancies, such as Hodgkin (HL) and non-Hodgkin lymphoma (NHL), acute (ALL) or chronic lymphocytic leukemia (CLL) [4].All tumor types treated with this therapy had a high initial complete response (CR) rate, but long-term disease-free survival can still be improved [4]The therapeutic success of CAR T cells is sometimes discrepant as it is shaped by several factors, boosting the conduction of a comparative analysis to address the global impact of in vivo and ex vivo conditions that influence CD19 CAR T cell performance in clinical trials.
Here, we analyzed the rates of the primary outcomedefined as best complete response (BCR) -and secondary outcomes defined as 12-month overall survival (OS) and best objective response (BOR) of CD19-positive leukemia or lymphoma patients treated with CD19 CAR T cells containing different hinge, transmembrane (TMD), and costimulatory domains.We have also analyzed the impact of different parameters related to CAR T cell manufacturing conditions, such as the type of interleukin used for CAR T cell expansion, CAR T cells activation method, and cell population transduced with the CAR.We have also evaluated the number of CAR T cell infusions, amount of CAR T cells injected/Kg, CD19 CAR type (name), tumor type, and age.This meta-analysis will be helpful as a hypothesis-generating instrument as it tries to capture effects in the literature that is still recent, lacking randomized clinical trials and large observational studies.

Search strategy
We accomplished a systematic review and meta-analysis according to the PRISMA statement [5,6], registered on PROSPERO (CRD42022360268).The main study question is the rate of BCR in patients undergoing treatment for B cell malignancies according to the CD19 CAR T cells hinge, transmembrane, and costimulatory domains.The MEDLINE/PubMed database was searched from the inception until August 2021, using the following keywords: "receptors, chimeric antigen"[MeSH Terms] OR ("receptors"[All Fields] AND "chimeric"[All Fields] AND "antigen"[All Fields]) OR "chimeric antigen receptors"[All Fields] OR ("chimeric"[All Fields] AND "antigen"[All Fields] AND "receptor"[All Fields]) OR "chimeric antigen receptor"[All Fields]) AND "CD19"[All Fields].

Study eligibility criteria
The inclusion criteria were patients with CD19-positive leukemia or lymphoma treated with second or third-generation CD19 CAR T cells.Only studies with original data and in English were included.Grey literature and reference lists from included studies were also considered.
The exclusion criteria were studies with (a) no primary outcome reported, (b) dual CAR, (c) other CAR cells types, such as CAR macrophages, (d) combinations with CAR T cells targeting other molecules or with other targeted or non-targeted therapies, such as hematopoietic stem cell transplant, (e) patients with multiple myeloma and other non-hematological tumors, (f ) case series, (g) studies such as meta-analyses, reviews, case reports, protocols, books, letters to the editor, comments or specialists' opinions, abstracts, and (h) pre-clinical studies.Studies ≤ 10 patients were included in the evidence summary but were excluded from the meta-analysis due to statistical constraints.

Data extraction
Data extracted comprised the rate of successful outcomes versus the sample included in the study, and BCR was defined as the primary outcome.The secondary outcomes were OS and BOR.For the meta-analysis, categorical covariates were the types of hinge, TM, and costimulatory (costimulation) domains in the CAR, CAR T cell manufacturing conditions, such as the interleukin used for CAR T cell expansion, CAR T cells activation method, and cell population transduced with the CAR -PBMCs or other specific subsets -(CAR T cell type), as well as the CD19 CAR type (CAR name), and tumor type.Numerical covariates were patient age, number of CAR T cells injected/Kg, and the number of CAR T cells infusions.
Two independent investigators (ERS and NSPC) screened titles and abstracts with ties resolved by a third person (VAP).Three authors (NSPC, VAP, GCPS) independently performed the full-text review and extracted the data, and ERS resolved disagreements.

Data syntheses
The data was presented in a summary of evidence and synthesized as forest plots, with studies ordered by publication year.All methodological details of the meta-analysis were included in the Supplementary Methods.

Risk of bias assessment
Risk of bias assessment adopted the Modified Institute of Health Economics Tool for bias analysis [7] and was performed independently by three authors (NSPC, VAP, GCPS).

Statistical analysis
Statistical analysis was performed with RStudio version 1.1.383(The R Foundation for Statistical Computing, Vienna, Austria), using meta and metafor packages [8,9].

Results
Fifty-six studies were included in the systematic review with a total of 3493 patients, 2904 treated with CAR T, and 2809 patients analyzed for rate estimation of BCR.Of these patients, 1440 presented a CR, and 1587 had an objective response (OR).We have also evaluated 12 months-OS, having 42 studies with a total of 2992 patients included, 2479 patients treated with CAR T, and 2393 patients analyzed, of whom 1567 were alive at 12 months.
A total of 46 studies with more than or equal to 10 patients were included in the meta-analysis involving 3421 patients, of whom 2837 were treated with CAR T and 2746 patients analyzed for rate estimation of the primary outcome BCR, being 1251 patients presenting CR and 1571 presenting OR, one of the secondary outcomes evaluated.For the other secondary outcome assessed, OS, we had 37 studies with 2949 patients, 2439 patients treated with CAR T, and 2356 patients analyzed for OS, of whom 1547 were alive at 12 months.The PRISM flow diagram is present in Fig. 1, and the summary of evidence in Table 1.

General clinical responses of CD19 CAR T therapy
The general proportion of BCR was 56% (95%CI: 49 -63%), the I 2 was 81%, and the τ 2 was 0.7911 indicating a large between-study variance (Fig. 2).However, it equals or exceeds 50% in 28 of 46 studies (Fig. 2).Table 2 summarizes meta-analysis data for primary outcome BCR (also presented in full version with references as Suppl.Table 1).The bias assessment is presented in Fig. 3.
The general proportion of OS was 60% (95%CI: 53 -67%), the I 2 was 87%, and the τ 2 was 0.5642 (Suppl.Figure 1 and Suppl.Table 2) indicating a moderate between study variance.The overall rate of BOR with CD19 CAR T therapy was 75% (95% CI: 68 -82%, I 2 = 78%) with a very high between-study variance (τ2 = 1.2262) and rates equal to or above 50% in 40 of 46 studies (Suppl.Figure 2 and Suppl.Table 3).Together, these data indicate substantial heterogeneity.The bias assessment for OS and BOR are also presented in Suppl.Figure 3.All the other forest plots are presented as Suppl.Figure 4 to 39.
The BCR (Suppl Fig. 10), OS (Suppl Fig. 11), and BOR (Suppl Fig. 12) proportions were similar for activation and expansion of CAR T cells with anti-CD3/CD28 beads or anti-CD3 mAb.Considering the cell population transduced with the CAR, we have found similar BCR (Suppl Fig. 13) and BOR (Suppl Fig. 14) rates when using full PBMCs or CD4/CD8 1:1, CD8 only, or other specific

Meta-regression
The meta-regression showed that the group aged above 18 presented a low but significant amount of heterogeneity explained by this variable (H 2 = 7.5535) and that the moderator is inversely related to BCR, suggesting that the effect size favors the younger patient (estimate= -1.3211; p = 0.005).Also, costimulation based on CD28 and third-generation CD28/4-1BB presents a small amount of heterogeneity explained (H 2 = 9.1079), but both were statistically significant moderators (p = 0.0391 and p = 0.0493, respectively).For BOR, the attributable heterogeneity for costimulatory domains was H 2 = 7.5535, and CD28 and 4-1BB were significant for this observation (p = 0.0047 and p = 0.0355).The attributable heterogeneity for the CAR T cell product was small (H 2 = 7.4956); however, there was an inverse effect for Tisa-cel and JCAR014 as moderators (p = 0.0336 and p = 0.0097).Finally, for OS, the attributable heterogeneity for the CAR T cell product was H 2 = 6.0343, and only the treatment with JCAR014 presented an inverse and statistically significant moderator effect (p = 0.0215).

Risk of bias assessment
A predominant low risk of bias was assessed for the primary and the secondary outcomes, presented in Suppl.[70].The difference in pooled BCR from these two studies compared with ours can be explained, at least in part, by the mixed tumor types included in our study, such as ALL, CLL, and other NHL subtypes.When we compared ALL and NHL in our sensitivity analysis for tumor type, the most expressive differences between them were also found for BCR (73 versus 51%), followed by BOR (80 versus 71%) rates, while both tumors resulted in similar OS rates (59 versus 57%).Our data also suggest that regardless of whether patients have had high objective responses or not, they might have survival benefits from CD19 CAR T therapy.Among the CAR T manufacturing conditions evaluated herein, the cell populations chosen to build the CAR product and the cytokine used for T cell expansion promoted the most relevant differences for the clinical outcomes analyzed, mainly for OS.PBMCs had higher OS but similar BOR and BCR rates compared to CD4/CD8 1:1 clustered with CD8 and other specific subsets for analysis.The clustering of CD4/CD8 1:1, CD8 alone, or others might have influenced the results obtained since there is pre-clinical and clinical evidence that CD4:CD8 1:1 seems to outperform other populations.However, we decided to cluster these groups due to the small number of clinical studies available to evaluate each one of these cell populations separately.ILs different from IL-2 used for CD19 CAR T cell manufacturing showed higher OS rates despite similar BCR and lower BOR, evidencing the necessity of running clinical trials using different cytokines for CAR T cell expansion to evaluate their impacts on clinical responses.The CAR T cell activation and expansion methods were equivalent for all outcomes evaluated.
Considering the covariate age, patients under 18 had notably higher BCR and BOR rates but similar OS compared to older patients.CD19 CAR T cell therapy is known to induce a high clinical response rate in children and young adults, especially with B-ALL, but relapses are still a current issue [62], explaining, at least in part, the similar OS despite the higher BCR rates found in younger patients.
Regarding the CAR T cell dose effect, higher BCR, BOR, and OS rates were found for patients treated with doses between 1 and 4.9 million cells/kg compared to those with doses greater than 100 million cells/kg.The dose-effect might be biased considering the higher BCR and BOR rates found for younger patients, usually treated with lower CAR T cell doses.Nevertheless, the age bias can be ruled out for the higher OS rates observed for lower CAR T doses since OS was not affected by age.When the number of CAR T infusions was evaluated, we noted that three or more infusions presented lower rates for the evaluated outcomes.This result is critical because higher CAR T doses with repeated infusions are known to enhance toxicity [71,72] despite the evident increased manufacturing cost.These results might affect the design of future comparative CD19 CAR T cells-based clinical trials, which can be focused on testing different dose scales up to 100 million cells/kg, limiting the administration to one or two infusions.
The comparison of different molecules used to build the structural CD19-directed CAR hinge (CD8, CD28, or IgG4), transmembrane (CD8 or CD28), and costimulatory domains (CD28 or 4-1BB) showed that the presence of CD28 in these three domains revealed higher rates for all the clinical outcomes evaluated.It might be possible that the different CAR domains act synergistically since they are part of the same functional full costimulatory molecule in human immune cells.However, we cannot affirm or discard this hypothesis based on our data.Particularly considering OS, the most relevant rate difference was found when CD28 was in the CAR's hinge and costimulatory regions.For BCR, the rate differences between CD28 and other molecules tested were less relevant.A CAR hinge and transmembrane-based comparison with clinical data has never been performed before in the literature, and our meta-analysis gives us some evidence that must be further investigated in future studies to clarify the possibility of synergism combining different/ equal domains.For the costimulatory domains, data recently published in a meta-analysis focused on patients with diffuse large B-cell lymphoma (DLBCL) treated with CD19 CAR T cells corroborated our findings, showing higher BCR and BOR rates of CD28 (57% BCR and 81% BOR) compared to 4-1BB (42% BCR and 70% BOR).However, they found a non-significant statistical difference between CD28 and 4-1BB considering the 12-month OS rate for DLBCL patients [69].In the same study, the CD28-based Axi-cel had higher rates for all outcomes evaluated compared with the 4-1BB Tisa-cel CAR T for the treatment of DLBCL patients, with a BCR rate of 57% versus 36%, OS rate of 65% versus 49%, and BOR rate of 82% versus 58%, respectively [69]A clinical trial comparing CD19 CAR-T containing either CD28 or 4-1BB was performed to treat ten ALL patients, five treated with each type of construction in a dose of 0.62 × 10 6 CAR T cells/kg.This study showed similar responses for both treatments, with the CD28 group resulting in 3 CR, 1 PR, and one no response (NR), and the 4-1BB with 3 CR, 0 PR, and 2 NR.Despite the superior number of NR patients in the 4-1BB group, this group had a unique patient with an ongoing anti-tumor response evaluated five months after treatment [73].This clinical trial was not conclusive due to the limited number of patients.Still considering the costimulatory domain of the CAR, Cappel and Kochenderfer recently reviewed and compared CAR T cell clinical studies based on different targets and having CD28 or 4-1BB as costimulatory domains, including but not limiting CD19 as a target.This general review showed that the available data from clinical trials do not demonstrate a clear advantage of either CD28-costimulated or 4-1BB-costimulated CARs for treating B cell lymphomas or B-ALL, pointing out that more extensive studies and comparative clinical trials must be performed to allow a conclusion about the performance of the different costimulatory domains against B-cell malignancies [74].
This study is the pioneer in evaluating the impact of the hinge and TMD CAR domains in addition to costimulatory domains in CD19 CAR T cell's clinical response for B cell leukemia and lymphoma, which is an essential unanswered question in the field.In summary, several covariates analyzed might have a positive impact on all the evaluated clinical outcomes BCR, OS, and BOR of patients treated with CD19 CAR T cell therapies, such as age inferior to 18 years old, injection of 1 to 4.9 million CAR T cells per kg, with one CAR T cell infusion -without discard a potential efficiency using two doses -and CD28 constituting the hinge, transmembrane, and costimulatory domains of the CAR, as in Axi-cel product, and must be better explored in future comparative clinical trials.
The lack of randomized trials or large observational studies on CAR T cells justifies the implementation of this meta-analysis, which intends to provide insights on the ongoing procedures for further research, raising questions and spotting potential aspects of interest in the current approaches.Due to the unavoidable heterogeneity observed, the results of this meta-analysis are not deemed for clinical decision-making but to improve the understanding of this complex and multifaceted treatment instead.The extrapolation and generalization of the results obtained in this meta-analysis should be made with caution since it may be biased by the different study designs and characteristics considering CAR structures, CAR T cell manufacture conditions, doses, tumor type, autologous cells isolated from each individual heavily pretreated, and other variables.

Fig. 2
Fig. 2 The Forest Plot represents the overall rate of the primary outcome Best Complete Response (BCR) of patients treated with CD19 CAR T therapy based on the studies included in the meta-analysis

Fig. 3
Fig. 3 Funnel, Baujat, and Radial plots showing the heterogeneity observed for the primary outcome Best Complete Response (BCR) of patients treated with CD19 CAR T therapy based on the studies included in the meta-analysis the manuscript; E.R.S. conceived the study, performed literature search, article selection, and manuscript writing and revision.Funding N.S.P.C had a Brazilian National Council for Scientific and Technological Development (CNPq) scholarship (143179/2021-7; 140514/2022-8).V.A.P was supported by a Federal University of ABC institutional scholarship (PIC-UFABC).E.R.S had a grant from Sao Paulo Research Foundation/Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil, number 2018/17656-0 and 2023/03631-3.

Table 1
Evidence summary
[69]68] 37,38,and 39, respectively.meta-analysis,therearealsosomeoutliers,such as one published byRamos et al. (2016), showing only 13% BCR and 19% BOR (N = 16 patients, no OS reported), that can be explained by the employment of a first-generation CAR, which usually fails to reach effective antitumor responses[67,68].For comparison, a meta-analysis focused on DLBCL conducted in 2022 by Ying and collaborators showed a similar pooled 63% OS rate and 74% BOR, diverging only by an expressively lower 48% BCR[69].Additionally, another meta-analysis published in 2021 by Aamir et al., focused on ALL patients, reported an 82% BCR rate.Neither OS nor BOR were reported in this study for comparison